Lysophosphatidic acids (LPAs) are bioactive phospholipids that stimulate cell proliferation, migration, platelet aggregation, and survival. LPAs influence many physiological processes, such as brain and vascular development. Aberrant LPA production, expression, and signalling have been linked to cancer initiation (e.g., tumorigenesis), progression, angiogenesis, and metastasis. LPAs are thought to play a role in a number of cancers, including ovarian cancer, breast cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, and multiple myeloma. LPAs also have been linked to cardiovascular disease (e.g., atherosclerosis, atherothrombosis), platelet aggregation, pulmonary inflammatory diseases, renal diseases, ischemia perfusion injury, wound healing, neuropathic pain, neuropsychiatric disorders, reproductive disorders, and fibrosis.
Liquid chromatography-mass spectrometry (LC-MS) is one of the most popular quantification methods for LPA, especially for individual LPA quantifications. A wide variety of methods for LPA sample preparation have been developed. Liquid-liquid extraction (LLE) is the common methodology for LPA enrichment for biological samples. For example, the LLE method developed by Bligh-Dyer (Can. J. of Biochemistry and Physiology, 1959, 37, 911-917) has been modified and used in many LPA studies. These modified methods that require one- or two-step extractions are easy and fast. However, they are also problematic, because abundant potential interferences, such as lysophosphatidylcholine (LPC), can be co-extracted with LPA and affect LPA quantifications.
For example, Zhao et al. (J. Chromatography B, 2009, 877:3739-3742) reported that lysophosphatidylcholine (LPC) and lysophosphatidylserine (LPS) could lose the choline or serine head group to artificially generate LPA-like signals in electrospray ionization tandem mass spectrometry (ESI-MS/MS) at the ion source. This fact may greatly affect LPA quantification since the biological concentration of LPC is about 300 μM, which is hundreds of times higher than LPA (less than 5 μM) in healthy people. Meanwhile, some phospholipids, such as glycerophosphocholines and lysophosphatidylcholines, may cause a matrix effect that suppresses or enhances ionization process, shifts the retention time, and/or elevates the baseline. These influences may greatly reduce the accuracy and reproducibility of LPA quantification by LC-MS.
Other quantification methods besides LC-MS may also be affected by the presence of interfering phospholipids in LPA samples because of the similarities between LPA and other phospholipids. For example, phosphatidic acid (PA) has the same head group as LPA; lysophosphatidylethanolamine (LPE), LPS, and LPC all have alkyl chains with the same or similar length as LPA. Thus, if the detection is based on phosphate group binding or deaggregation by the LPA alkyl chain, the phospholipids that co-extracted with LPA from plasma would interfere with the detection and lead to a false positive result. Thus, removal of interferences is critical for an accurate quantification of LPA.